High CD46 Receptor Density Determines Preferential Killing of Tumor Cells by Oncolytic Measles Virus
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[CANCER RESEARCH 64, 4919–4926, July 15, 2004] High CD46 Receptor Density Determines Preferential Killing of Tumor Cells by Oncolytic Measles Virus Bambi D. Anderson,1,2 Takafumi Nakamura,1 Stephen J. Russell,1,3 and Kah-Whye Peng1,2 1Molecular Medicine Program, 2Mayo Clinic Cancer Center, and 3Department of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota ABSTRACT fects present in tumor cells and not in normal cells (8). One of the most important defense mechanism a cell has against viral infection is Live attenuated Edmonston B strain of measles virus (MV-Edm) is a induction of IFN-␣/ and IFN-inducible proteins, resulting in sup- potent and specific oncolytic agent, but the mechanism underlying its pression of protein synthesis and establishment of an antiviral state (9, tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell 10). However, viruses have evolved diverse strategies to evade or killing in normal cells. The CPE is dependent on expression of viral antagonize the IFN antiviral response (11). Thus, measles virus (MV) proteins and the presence of CD46, the major cellular receptor of MV- encodes the V and C accessory proteins that block IFN-␣/ produc- Edm. Using a virally encoded soluble marker peptide to provide a quan- tion and/or signaling, allowing the virus to replicate in the host cell titative readout of the level of viral gene expression, we determined that (12–15). The mechanism underlying MV-C inhibition of IFN-␣/ tumor cells and normal cells expressed comparable levels of viral proteins. signaling remains unclear (15), but the MV-V protein blocks the IFN CD46 mediates virus attachment, entry, and virus-induced cell-to-cell response by inhibiting phosphorylation of signal transducers and fusion. Using engineered cells expressing a range of CD46 densities, we activators of transcription 1 and 2 proteins (13). determined that whereas virus entry increased progressively with CD46 MV enters cells by binding via its hemagglutinin (H) attachment density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well protein to one of two cellular receptors, CD46 (16, 17) or signaling established that tumor cells express abundant CD46 receptors on their lymphocyte activation molecule. The pathogenic wild-type MV surfaces compared with their normal counterparts. Thus, at low CD46 (which is not selectively oncolytic) uses primarily signaling lympho- densities typical of normal cells, infection occurs, but intercellular fusion cyte activation molecule, expressed on activated T cells, B cells, and is negligible. At higher densities typical of tumor cells, infection leads to monocytes/macrophages, as a receptor (18, 19). In contrast, attenuated extensive cell fusion. Intercellular fusion also results in enhancement of vaccine strains such as MV-Edm use predominantly CD46 (20), viral gene expression through recruitment of neighboring uninfected cells which is ubiquitously expressed (usually at low density) by all human into the syncytium, further amplifying the CPE. Discrimination between cells except erythrocytes (21). In addition, CD46 is required to me- high and low CD46 receptor density provides a compelling basis for the diate intercellular fusion. Virally infected cells expressing the MV oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent. envelope glycoproteins, hemagglutinin (H) and fusion (F), on their cell surfaces fuse with neighboring cells through CD46 to form multinucleated syncytia, the characteristic CPE of MV-Edm infection. INTRODUCTION CD46, also known as membrane cofactor protein, plays an impor- tant role in protecting autologous cells from complement attack by Live attenuated Edmonston B strain of measles virus (MV-Edm) serving as a cofactor for Factor I-mediated inactivation of C3b and has potent and specific oncolytic activity against a variety of human C4b, thus blocking the complement cascade at the C3 activation stage tumors, including lymphoma, multiple myeloma, epithelial ovarian (22, 23). Indeed, CD46 is frequently overexpressed on cancer cells cancer, and glioma (1–6). MVCEA, a recombinant MV-Edm genet- compared with their normal counterparts, possibly as a mechanism for ically modified to express human carcinoembryonic antigen (CEA) as cancer cells to overcome lysis by complement (24). Overexpression of a biologically inert soluble marker for noninvasive monitoring of the CD46 and other membrane complement regulatory proteins, CD55 profiles of viral gene expression, is being tested in a Phase I clinical and CD59, has been documented in leukemias and gastrointestinal, trial for patients with recurrent epithelial ovarian cancer (7). hepatocellular, colorectal, endometrial, cervical, ovarian, breast, renal, MV-Edm is selectively oncolytic, causing extensive syncytium and lung carcinomas and found to limit the therapeutic potential of formation and cell killing in a variety of human tumor cells but monoclonal antibody therapy (3, 24). Overexpressed complement minimal cytopathic damage in nontransformed cells such as normal regulatory proteins have also been studied as potential targets for dermal fibroblasts, ovarian surface epithelium, mesothelial cells from cancer therapy using bispecific antibodies and anti-idiotypic vaccina- the peritoneal cavity, and peripheral blood lymphocytes (2, 3). Until tion (25). now, there have been no published studies addressing the mechanisms CPEs induced in MV-Edm-infected cells are dependent on virus underlying the tumor specificity of MV-Edm. However, elucidation of entry, expression of MV-H and MV-F, and the CD46 cellular recep- these mechanisms will be pivotal to future development of MV-Edm tor. We first quantitated the relative expression levels of viral proteins as a cancer therapeutic agent and may provide clues that can poten- in infected tumor cells versus nontransformed cells and found com- tially increase the efficacy or safety of this agent in the clinic. parable levels of gene expression despite striking differences in CPEs. A number of the oncolytic viruses currently being developed for The logical hypothesis to explain these observations was that there cancer therapy are tumor selective because they exploit genetic de- may be a correlation between CD46 receptor density and the strik- ingly different CPEs observed in tumor cells versus nontransformed Received 3/11/04; revised 5/4/04; accepted 5/12/04. Grant support: The Oliver S. and Jennie R. Donaldson Charitable Trust (B. D. cells on MV-Edm infection. To define the role of CD46 receptor Anderson), Mayo Foundation, Harold W. Siebens Foundation, George W. Eisenberg density in MV-Edm infection, a panel of Chinese hamster ovary Foundation, and NIH Grants CA100634-01 and HL66958-03P4. (CHO) clones expressing a range of surface densities of CD46 was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with generated. Rodent cells lack CD46, but if they are engineered to 18 U.S.C. Section 1734 solely to indicate this fact. express human CD46, they become infectable by MV-Edm and are Requests for reprints: Kah-Whye Peng, Molecular Medicine Program, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-8357; Fax: (507) 284-8388; E-mail: susceptible to the CPEs of MV-Edm (26). Using these CHO-CD46 [email protected]. transfectants, MV-eGFP [a recombinant MV-Edm expressing green 4919 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. HIGH CD46 RECEPTOR DENSITY DETERMINES TUMOR CELL KILL fluorescent protein (GFP)], and an adenoviral vector expressing MV- brief centrifugation step and frozen in liquid nitrogen before storage at Ϫ80°C. H/F proteins, we explored the relationship between CD46 receptor Virus titers were obtained by titration on Vero cells and expressed as 50% density, virus entry, and cell fusion. Virus entry increased progres- tissue culture infectious dose (TCID50)/ml. sively with CD46 receptor density and was quite efficient even at Infection Assays. The panel of human tumor and normal cells was infected ϭ relatively low receptor densities. In contrast, syncytium formation and with MV-CEA (MOI 0.2) for2hat37°C, after which the virus inoculum was removed, and the cells were maintained in standard medium for 48 h. The cell killing were minimal at low CD46 densities but increased rapidly cells were fixed with 0.5% glutaldehyde and stained with 0.2% crystal violet above a threshold CD46 expression level. These findings suggest that solution, and the CPEs were photographed. To quantitate the relative levels of CD46 is an interesting new cancer target and that the differential viral gene expression, the cells were infected with MV-CEA (MOI ϭ 0.4) or expression of CD46 in tumor cells versus normal cells dramatically MV-eGFP (MOI ϭ 0.4) and maintained in the presence of FIP to allow increases the susceptibility of tumor cells to the oncolytic activity of analysis of single infected cells by flow cytometry. Forty-eight h later, the MV-Edm, providing a mechanistic basis for tumor specificity of media were harvested, the number of viable cells per well was counted by MV-Edm. trypan blue exclusion, and the percentage of infected GFP-positive cells was analyzed by flow cytometry. The amount of virally encoded CEA marker MATERIALS AND METHODS peptide in the medium was analyzed by the Mayo Central Clinical Laboratory. CHO or CHO-CD46 cells were plated overnight (1 ϫ 105 cells/well) in a Cell Lines. African green monkey kidney Vero cells and human ovarian 12-well plate and infected the next day with MV-eGFP (MOI ϭ 0.5) or Ad5/35 carcinoma SKOV3ip.1, fibrosarcoma HT1080, and epithelial lung carcinoma (MOI ϭ 100) for2hat37°C. The cells were maintained in 10% fetal bovine A549 cells were maintained in DMEM (BioWhittaker, Walkersville, MD) sup- serum-DMEM (Ad5/35) or in medium containing FIP (80 nM).